The present invention relates to mass spectrometry of a solution, and particularly to a mass spectrometer for analyzing substances in a solution and an apparatus for combining a liquid chromatograph used for separation and analysis of a mixed sample and the mass spectrometer.
At present, development of mass spectrometry of biological substances is regarded as important in the field of analysis. Biological substances are generally dissolved in a solution as a mixture, so that an apparatus for combining a means for separating a mixture and a mass spectrometer is under development. As a typical apparatus of this method, there is a liquid chromatograph-mass spectrometer (hereinafter abbreviated to LC/MS) available. A liquid chromatograph (hereinafter abbreviated to LC) is superior in separation of a mixture but cannot identify each substance, whereas a mass spectrometer hereinafter abbreviated to MS) is highly sensitive and superior in identification ability but not suitable to analysis of a mixture. Therefore, an LC/MS using an MS as an LC detector is very useful in analysis of a mixture.
The LC/MS using the conventional atmospheric pressure chemical ionization method which is disclosed in Japanese Patent Application Laid-Open 5-325882 will be explained hereunder by referring to FIG. 9.
A sample solution eluted from a liquid chromatograph 14 is introduced into a metallic tube 3 via a pipe 1 and a connector 2. The sample solution comprises a sample and a mobile phase (a buffer solution which flows into the separation column when the sample is separated in the LC). However, for the purpose of improving the ionization efficiency in the ion source (according to the present invention, the term xe2x80x9cion sourcexe2x80x9d means a portion for converting a substance to be analyzed existing in the liquid phase to ions in the gas phase and includes a spray portion for spraying a solution, a vaporization portion for vaporizing droplets generated by the spray portion, and an ionization portion for ionizing a substance), a solvent different from the mobile phase may be added. The metallic tube 3 is embedded in a metallic block 4a. When the metallic block 4a is heated by a heating means such as a heater, a sample solution introduced in the metallic tube 3 is sprayed. Fine droplets generated by spraying are introduced into and vaporized in a vaporization portion 5 comprising a heated metallic block 4b. 
Sample molecules vaporized in the vaporization portion 5 are introduced into the ionization portion 6. A needle electrode 7 is installed in the ionization portion 6. When a high voltage is applied to this needle electrode 7 from a high voltage source 8a, a corona discharge is generated in the ionization portion 6.
Assuming A to be sample molecules to be analyzed and B to be molecules of a reaction gas, the atmospheric pressure chemical ionization method is a method for ionizing A by a chemical reaction of A and B. As a typical ion chemical reaction, there are a protonation reaction and a deprotonation reaction available as shown below.
A+BH30xe2x86x92AH+B (Protonation reaction)
A+Bxe2x88x92xe2x86x92(Axe2x88x92H)xe2x88x92+BH (Deprotonation reaction)
According to the prior art shown in FIG. 9, hydronium ions (H3O+) are generated when a corona discharge is generated in the atmosphere and ions Ali+ of the sample A are generated by using the following reaction between the hydronium ions and the sample molecules A.
A+H3O+xe2x86x92AH++H2O
Ions of the sample generated by chemical ionization in the ionization portion 6 have their trajectory deflected by a voltage applied to a deflection electrode 31 by a power source 30 and drift toward an ion introduction aperture 9a. The ions pass through the ion introduction aperture 9a and are introduced into a high vacuum portion 12 which is exhausted to a high vacuum by an exhaust system 10b via a differential pumping portion 11 which is evacuated by an exhaust system 10a and an ion introduction aperture 9b. When ions and solvent molecules pass through the ion introduction apertures 9a and 9b, they are cooled by adiabatic expansion, so that so-called clustering for condensing the ions and solvent molecules again occurs. To prevent the clustering, electrodes in which the ion introduction apertures 9a and 9b are formed are heated. The mass of ions introduced into the high vacuum portion 12 is analyzed by a mass spectrometric portion 13. Nonvolatile compounds that are not ionized in the ionization portion 6 are diffused in the atmosphere and captured by a capture plate 32.
An LC/MS using the conventional electrospray method which is disclosed in Japanese Patent Application Laid-Open 6-102246 will be explained by referring to FIG. 10. A sample solution eluted from the LC is introduced into the metallic tube 3 via the pipe 1 and the connector 2. A high voltage is applied between the metallic tube 3 and an electrode 21c in which the ion introduction aperture 9a is formed by using a high voltage source 8b and the sample solution is electrostatically sprayed. To assist electrospray, gas such as nitrogen gas is let flow from a spray gas outlet 40. When fine charged droplets generated by electrospray are vaporized, gaseous ions are generated. However, the diameter of droplets at the center of the jet is large and it is difficult to vaporize droplets with a large diameter, and furthermore when droplets with a large diameter adhere to the electrode 21c, the temperature of the electrode 21c drops and the ion intensity obtained in the mass spectrometric portion may vary. Therefore, a shielding plate 41 for shielding the center of the jet is installed between the metallic tube 3 and the electrode 21c and the outer periphery of the jet is sprayed toward the ion introduction aperture 9a. Generated ions are introduced into the high vacuum portion 12 via the ion introduction aperture 9a, the differential pumping portion 11, and the ion introduction aperture 9b and analyzed by the mass spectrometric portion installed in the high vacuum portion 12.
For analysis of biological substances and environmental contaminants, a method for analyzing a sample solution containing nonvolatile compounds of high concentration is required.
For example, in the LC, the mobile phase including a nonvolatile salt is often used experientially so as to enhance the separation ability and the reproducibility of the retention time. As a result, a method for analyzing a sample solution containing a nonvolatile salt is desirable for a detector of the LC.
Nonvolatile compounds are contained not only in samples obtained from a living organism such as urine, perspiration, and blood, but also is samples relating to the environment such as effluents from a factory and water of a lake or marsh. To remove nonvolatile compounds from these samples, a complicated pretreatment such as desalting is required. Therefore, in order to analyze biological substances and environmental contaminants quickly, a method for analyzing a sample solution containing nonvolatile compounds is required.
However, in a mass spectrometric apparatus using the conventional atmospheric pressure chemical ionization method shown in FIG. 9, when a sample solution containing nonvolatile compounds of high concentration is introduced into the ion source of the mass spectrometric apparatus comprising an ion source, a differential pumping portion, and a mass spectrometric portion, a problem arises that ions of a substance to be analyzed cannot be analyzed stably for many hours. The reason is that since the sample solution is sprayed by using the heated metallic tube, solvent molecules are vaporized in the tube and nonvolatile compounds are salted out on the inner wall of the tube. The inner diameter of the metallic tube becomes smaller because nonvolatile compounds are salted out on the tube wall, and the metallic tube finally clogs up. Therefore, the spray status varies with time, so that the ion generation in the ionization portion is adversely affected.
When fine droplets containing nonvolatile compounds adhere to the neighborhood of the ion introduction aperture installed between the ion source and the differential pumping portion, solvent molecules are vaporized and nonvolatile compounds are salted out around the aperture. In the conventional apparatus shown in FIG. 9, by installing the capture plate on the ion source side, a method for salting out nonvolatile compounds contained in droplets adhered to the capture plate on the capture plate is used so as to reduce salting out of nonvolatile compounds around the aperture. However, it is actually difficult to capture all droplets sprayed in the atmosphere on the capture plate and some of the droplets are diffused in the atmosphere and reach the aperture. Therefore, if the analysis is continued for many hours, an actual problem arises that salted nonvolatile compounds clog up the aperture and ions cannot be introduced into the mass spectrometric portion.
As an example of a sample solution containing nonvolatile compounds, a case where a sodium phosphate water solution (hereinafter described as a phosphoric acid buffer) having a concentration of 20 millimoles/liter is introduced into the ion source at a flow rate of 50 microliters per minute will be described. A phosphoric acid buffer is a mobile phase which is often used in a detector other than the MS, for example, in an LC having an ultraviolet absorption detector. When a conventional LC/MS uses a phosphoric acid buffer, the observed ion intensity starts dropping within about 30 minutes after the start of analysis and drops down to about {fraction (1/10)} of the initial value after one hour has elapsed, and the continuation of analysis becomes difficult.
Whether a problem that the metallic tube or aperture clogs tip due to salting out of nonvolatile substances arises or not depends on the kind of nonvolatile substances and the total amount of nonvolatile substances (namely, the concentration of nonvolatile substances and the flow rate of the sample solution) introduced into the ion source. For example, when substances are analyzed at a flow rate of 1 microliter per minute continuously for several hours using a phosphoric acid buffer having a concentration of 10 millimoles/liter, the ion intensity observed by the mass spectrometer may decrease.
Even in the apparatus using the conventional electrospray method shown in FIG. 10, the outer periphery of a jet is sprayed toward the ion introduction aperture, so that some of the droplets generated by the spray reach the neighborhood of the ion introduction aperture. Therefore, when a sample solution containing nonvolatile compounds of high concentration is introduced into the ion source, there is a possibility that the ion introduction aperture may clog up with nonvolatile compounds salted out by vaporization of droplets. Therefore, in the same way as with an apparatus using the atmospheric pressure chemical ionization method shown in FIG. 9, a problem arises that if the analysis is continued for many hours, the aperture clogs up and ions cannot be introduced into the mass spectrometric portion.
For the aforementioned reason, an apparatus having an ion source allowing stable analysis for many hours even if a solution containing nonvolatile compounds of high concentration is introduced is required.
An object of the present invention is to prevent a metallic tube and an ion introduction aperture from clogging up by salting out of nonvolatile compounds. Another object of the present invention is to allow analysis by an LC/MS using a mobile phase containing a nonvolatile salt which cannot be used for many hours conventionally.
According to the present invention, the above objects are accomplished by introducing a sample solution containing nonvolatile compounds sent to the ion source into the metallic tube, spraying the sample solution by electrospray at a high voltage applied between the metallic tube and the electrode externally installed, ionizing gaseous sample molecules obtained by vaporizing obtained droplets by chemical reaction, and analyzing ions of sample molecules by the MS.
Namely, the present invention is characterized in the method and constitution that nonvolatile compounds contained in a sample solution are ionized by the first ionization means and removed by the electric field, and then a substance contained in the sample solution to be analyzed is ionized by the second ionization means, and ions of the target substance are analyzed by the spectrometric portion.
Since a sample solution sent to the ion source is sprayed by electrospray, no nonvolatile compounds will be salted out by vaporization of solvent molecules and the metallic tube can be prevented from clogging.
Electrospray is a method for collecting ions in a solution on the liquid surface by the electric field and crushing the solution by the force of repulsion acting between ions so as to generate fine charged droplets. When charged droplets obtained by electrospray are vaporized, ions existing in the solution can be fetched in the gas phase. Therefore, some of nonvolatile compounds which exist in the solution as ions can be converted to gaseous ions by electrospray. Ions generated by electrospray are drifted by the electric field between the metallic tube and the metallic block constituting the vaporization portion and captured by the metallic block or the trajectory thereof is curved by the potential applied to the needle electrode installed in the ionization portion, so that they cannot reach the ion introduction aperture. Therefore, no nonvolatile compounds will salt out around the ion introduction aperture and the aperture will not be clogged out.
A substance to be analyzed which is not ionized in the solution is not converted to gaseous ions by electrospray but diffused in the atmosphere as gaseous molecules which are electrically neutral and reaches the ionization portion. In the ionization portion, ions of the substance to be analyzed are generated by a corona discharge generated by the needle electrode and chemical ionization reaction caused by it. Ions of the substance to be analyzed are fetched and analyzed by the mass spectrometric portion in a vacuum via the ion introduction aperture.
A nonvolatile salt which is represented by sodium phosphate is dissociated in a solvent such as water and exists as ions. Therefore, the LC/MS of the present invention can use a mobile phase containing a nonvolatile salt which is represented by a phosphoric acid buffer though it is conventionally difficult to use for many hours.